Orthotopic liver transplantation is the only available treatment for severe liver failure, but it is currently limited by organ shortage. One technical challenge that has thus far limited the ...development of a tissue-engineered liver graft is oxygen and nutrient transport. Here we demonstrate a novel approach to generate transplantable liver grafts using decellularized liver matrix. The decellularization process preserves the structural and functional characteristics of the native microvascular network, allowing efficient recellularization of the liver matrix with adult hepatocytes and subsequent perfusion for in vitro culture. The recellularized graft supports liver-specific function including albumin secretion, urea synthesis and cytochrome P450 expression at comparable levels to normal liver in vitro. The recellularized liver grafts can be transplanted into rats, supporting hepatocyte survival and function with minimal ischemic damage. These results provide a proof of principle for the generation of a transplantable liver graft as a potential treatment for liver disease.
Celotno besedilo
Dostopno za:
DOBA, IJS, IZUM, KILJ, NUK, PILJ, PNG, SAZU, UILJ, UKNU, UL, UM, UPUK
The only treatment available for end stage liver diseases is orthotopic liver transplantation. Although there is a big donor scarcity, many donor livers are discarded as they do not qualify for ...transplantation. Alternatively, decellularization of discarded livers can potentially render them transplantable upon recellularization and functional testing. The success of this approach will heavily depend on the quality of decellularized scaffolds which might show variability due to factors including age. Here we assessed the age-dependent differences in liver extracellular matrix (ECM) using rat and human livers. We show that the liver matrix has higher collagen and glycosaminoglycan content and a lower growth factor content with age. Importantly, these changes lead to deterioration in primary hepatocyte function potentially due to ECM stiffening and integrin-dependent signal transduction. Overall, we show that ECM changes with age and these changes significantly affect cell function thus donor age should be considered as an important factor for bioengineering liver substitutes.
The realization of long-term human organ preservation will have groundbreaking effects on the current practice of transplantation. Herein we present a new technique based on subzero nonfreezing ...preservation and extracorporeal machine perfusion that allows transplantation of rat livers preserved for up to four days, thereby tripling the viable preservation duration.
Celotno besedilo
Dostopno za:
DOBA, IJS, IZUM, KILJ, NUK, PILJ, PNG, SAZU, UILJ, UKNU, UL, UM, UPUK
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Engineering of functional vascularized liver tissues holds great promise in addressing donor organ shortage for transplantation. Whole organ decellularization is a cell removal method ...that retains the native vascular structures of the organ such that it can be anastomosed with the recipient circulation after recellularization with healthy cells. However, a main hurdle to successful implantation of bioengineered organ is the inability to efficiently re-endothelialize the vasculature with a functional endothelium, resulting in blood clotting which is the primary cause of failure in early transplant studies. Here, we present an efficient approach for enhancing re-endothelialization of decellularized rat liver scaffolds by conjugating the REDV cell-binding domain to improve attachment of endothelial cells (EC) on vascular wall surfaces. In order to facilitate expression and purification of the peptide, REDV was fused with elastin-like peptide (ELP) that confers thermally triggered aggregation behavior to the fusion protein. After validating the adhesive properties of the REDV-ELP peptide, we covalently coupled REDV-ELP to the blood vasculature of decellularized rat livers and seeded EC using perfusion of the portal vein. We showed that REDV-ELP increased cell attachment, spreading and proliferation of EC within the construct resulting in uniform endothelial lining of the scaffold vasculature. We further observed that REDV-ELP conjugation dramatically reduced platelet adhesion and activation. Altogether, our results demonstrate that this method allowed functional re-endothelialization of liver scaffold and show great potential toward the generation of functional bioengineered liver for long-term transplantation.
There is a critical need for novel organ replacement therapies as the grafts for transplantation fall short of demand. Recent advances in tissue engineering, through the use of decellularized scaffolds, have opened the possibility that engineered grafts could be used as substitutes for donor livers. However, successful implantation has been challenged by the inability to create a functional vasculature. Our research study reports a new strategy to increase efficiency of endothelialization by increasing the affinity of the vascular matrix for endothelial cells. We functionalized decellularized liver scaffold using elastin-like peptides grafted with REDV cell binding domain. We showed that REDV-ELP conjugation improve endothelial cell attachment and proliferation within the scaffold, demonstrating the feasibility of re-endothelializing a whole liver vasculature using our technique.
Liver tissue engineering has emerged as a promising approach in organ transplantation but has been hampered by the lack of a reliable and readily available cell source. Human induced pluripotent stem ...cells hiPSCs have been highlighted as a desirable source, due to their differentiation potential, ability to self‐renew, and the possibility of making patient‐specific cells. We developed a decellularization protocol that efficiently removes cellular material while retaining extracellular matrix components. Subsequently, hiPSCs were differentiated on decellularized human liver extracellular matrix (hDLM) scaffolds using an established hepatic differentiation protocol. We demonstrate that using hDLM leads to upregulation markers of hepatic functions when compared with standard differentiation conditions. In addition, expression of a number of hepatic transcription and nuclear factors were found to be within levels comparable with those of primary human adult hepatocytes. Analysis of progression of differentiation on hDLM demonstrated that hepatic developmental marker expression was consistent with hepatic development. The hDLM‐derived cells exhibited key hepatic characteristics that were comparable with those observed in primary neonatal human hepatocytes. We investigated the optimal timing of the introduction of hDLM into the differentiation protocol and found that the best results are obtained when cells are plated on hDLM since the earliest stages and accompanied by a progressive loss of sensitivity to substrate composition at later stages. The significance of this work is that it allows for the development of differentiation protocols that take into account signals from extracellular matrix, closely recapitulating of the in vivo micro‐environment and resulting in cells that are phenotypically closer to mature hepatocytes.
Growth factor therapy is a promising approach for chronic diabetic wounds, but strategies to efficiently and cost-effectively deliver active molecules to the highly proteolytic wound environment ...remain as major obstacles. Here, we re-engineered keratinocyte growth factor (KGF) and the cellular protective peptide ARA290 into a protein polymer suspension with the purpose of increasing their proteolytic resistance, thus their activity in vivo. KGF and ARA290 were fused with elastin-like peptide (ELP), a protein polymer derived from tropoelastin, that confers the ability to separate into a colloidal suspension of liquid-like coacervates. ELP fusion did not diminish peptides activities as demonstrated by ability of KGF-ELP to accelerate keratinocyte proliferation and migration, and ARA290-ELP to protect cells from apoptosis. We examined the healing effect of ARA290-ELP and KGF-ELP alone or in combination, in a full-thickness diabetic wound model. In this model, ARA290-ELP was found to accelerate healing, notably by increasing angiogenesis in the wound bed. We further showed that co-delivery of ARA290 and KGF, with the 1:4 KGF-ELP to ARA290-ELP ratio, was the most effective wound treatment with the fastest healing rate, the thicker granulation tissue and regenerated epidermis after 28 days. Overall, this study shows that ARA290-ELP and KGF-ELP constitute promising new therapeutics for treatment of chronic wounds.
Irreversible electroporation (IRE) is an emerging, minimally invasive technique for solid tumors ablation, under clinical investigation for cancer therapy. IRE affects only the cell membrane, killing ...cells while preserving the extracellular matrix structure. Current reports indicate tumors recurrence rate after IRE averaging 31% of the cases, of which 10% are local recurrences. The mechanisms for these recurrences are not known and new explanations for incomplete cell death are needed. Using finite elements method for electric field distribution, we show that presence of vascular structures with blood leads to the redistribution of electric fields leading to the areas with more than 60% reduced electric field strength in proximity to large blood vessels and clustered vessel structures. In an in vivo rat model of liver IRE ablation, we show that cells located in the proximity of larger vessel structures and in proximity of clustered vessel structures appear less affected by IRE ablation than cells in the tissue parenchyma or in the proximity of small, more isolated vessels. These findings suggest a role for "electric field sinks" in local tumors recurrences after IRE and emphasize the importance of the precise mapping of the targeted organ structure and conductivity for planning of electroporation procedures.
Initially hailed as the ultimate solution to organ failure, engineering of vascularized tissues such as the liver has stalled because of the need for a well-structured circulatory system that can ...maintain the cells seeded inside the construct. A new approach has evolved to overcome this obstacle. Whole-organ decellularization is a method that retains most of the native vascular structures of the organ, providing microcirculatory support and structure, which can be anastomosed with the recipient circulation. The technique was first applied to the heart and then adapted for the liver. Several studies have shown that cells can be eliminated, the extracellular matrix and vasculature are reasonably preserved and, after repopulation with hepatocytes, these grafts can perform hepatic functions in vitro and in vivo. Progress is rapidly being made as researchers are addressing several key challenges to whole-organ tissue engineering, such as ensuring correct cell distribution, nonparenchymal cell seeding, blood compatibility, immunological concerns, and the source of cells and matrices.
Supercooling preservation holds the potential to drastically extend the preservation time of organs, tissues and engineered tissue products, and fragile cell types that do not lend themselves well to ...cryopreservation or vitrification. Here, we investigate the effects of supercooling preservation (SCP at -4(o)C) on primary rat hepatocytes stored in cryovials and compare its success (high viability and good functional characteristics) to that of static cold storage (CS at +4(o)C) and cryopreservation. We consider two prominent preservation solutions a) Hypothermosol (HTS-FRS) and b) University of Wisconsin solution (UW) and a range of preservation temperatures (-4 to -10 (o)C). We find that there exists an optimum temperature (-4(o)C) for SCP of rat hepatocytes which yields the highest viability; at this temperature HTS-FRS significantly outperforms UW solution in terms of viability and functional characteristics (secretions and enzymatic activity in suspension and plate culture). With the HTS-FRS solution we show that the cells can be stored for up to a week with high viability (~56%); moreover we also show that the preservation can be performed in large batches (50 million cells) with equal or better viability and no loss of functionality as compared to smaller batches (1.5 million cells) performed in cryovials.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Severe liver disease is the 12th leading cause of death in the USA, with organ transplantation often being the only viable option for treatment. However, due to the shortage of viable donor livers, ...it is estimated that over 1200 patients died in 2015 while waiting for liver transplantation. This highlights the need for alternative sources of viable organs. In this study, we describe a method that provides the groundwork for the development of functional liver grafts. The approach described here is for removal of cells from intact livers and subsequently repopulating them with functional liver cells. Briefly, rat livers are harvested and subjected to a series of perfusion decellularization steps using an anionic detergent such that an intact decellularized liver matrix (DLM) scaffold with preserved vascular architecture is obtained. Further, we describe methods to recellularize DLM scaffolds with adult primary hepatocytes, creating a liver graft that exhibits hepatic functions in vitro.
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